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Bioluminescence: Investigating Glow-in-the-Dark Dinoflagellates
Abstract Imagine seeing waves glowing a beautiful blue color. The marine dinoflagellate Pyrocystis lunula is responsible for this magnificent phenomenon. Pyrocystis lunula''is a bioluminescent organism—bioluminescence is the production of light by living organisms. But does this organism always glow, no matter what the conditions, such as how much light there is? In this biotechnology science fair project, you will investigate how altering this dinoflagellate's exposure to light and dark affects its bioluminescence. Objective The objective of this biotechnology science fair project is to investigate how the bioluminescence of the marine dinoflagellate ''Pyrocystis lunula is affected by changes to its light-dark cycle. Introduction Marine dinoflagellates are the main contributors to a phenomenon commonly known as phosphorescence of the sea. When the concentration of these'bioluminescent' organisms in the water near shore is high, the wave crests glow with a luminous blue light (see Figure 1, below). Wet sand on the beach even glows blue when you step on it! The reason that the light appears on wave crests and in the sand near your feet is that the bioluminescent organisms glow when they are subjected to mechanical stress. They sense that they are being pushed and pulled in the waves, and in the sand near your feet, and respond by producing the light. It is not clear what sort of adaptive advantage this light might have for the organisms, but it makes for a beautiful show if you are lucky enough to witness it. There are many examples of bioluminescence in nature, with the most familiar being the firefly. Bioluminescence evolved independently in many different organisms. The biochemical basis for bioluminescence is the luciferin-luciferase reaction, shown in Figure 2, below. In this reaction, the enzyme luciferase'oxidizes' the substrate luciferin to convert chemical energy into light energy. Luciferase is an enzyme, a type of protein that speeds up chemical reactions. The reaction that luciferase catalyzes is the oxidation of luciferin by molecular oxygen (O2) to form oxyluciferin plus light. See The Bioluminescence Web Page from the University of California, Santa Barbara, listed in the Bibliography, for an animated diagram showing this reaction. This biotechnology science fair project investigates the response of the bioluminescent system of the dinoflagellate Pyrocystis lunula (P. lunula) to changes in its light-dark cycle (you can also use Pyrocystis fusiformis). P. Lunula are tiny plants that live in the ocean. They are unicellular algae that look like delicate, golden-green eyes when magnified, and produce oxygen and sugars, like all plants do. P. lunula sets its bioluminescence by a biological clock. In the dark, the cells produce the chemicals that are required for the luciferin-luciferase reaction. Both photosynthesis and bioluminescence are controlled by circadian cycles in''Pyrocystis lunula,'' and in various other dinoflagellates. You can obtain P. lunula and P. fusiformis as cultures of the cells in seawater. You do not need to feed them since they use ambient light for photosynthesis, producing their own oxygen and food. The cultures can last several weeks to months. They need light to grow, and prefer to remain between 50°F and 70°F. Terms and Concepts *Marine dinoflagellate *Phosphorescence of the sea *Bioluminescence *Mechanical stress *Luciferin-luciferase reaction *Oxidize *Substrate *Enzyme *Catalyze *''Pyrocystis lunula'' *Algae *Biological clock *Circadian cycle *Cell culture *Scintillon Questions *What other sorts of stress cause P. lunula cells to emit light? *Based on your research, what is the "burglar alarm" hypothesis for why dinoflagellates glow in turbulent water? *What is the biological classification (kingdom, phylum, class, order, family, etc.) for Pyrocystis lunula? *How are dinoflagellates related to the algae? *What is the chemical structure for luciferin? *What is the chemical equation for the luciferin-luciferase reaction? *What are some examples of circadian cycles in marine organisms? *What is the definition of an enzyme? *What is the definition of a substrate? *What is the name of the structure in dinoflagellates that produces bioluminescent light? Materials and Equipment Note: You can use P. lunula or P. fusiformis for this science fair project. The cells can be obtained from the sources below: *If you are using: *#''Pyrocystis lunula,'' cells are available from the University of Texas, Austin, which maintains a culture collection of algae. The website ishttp://www.sbs.utexas.edu/utex/teachingKits-bioluminescence.aspx. The cultures are delivered in 6 glass test tubes. *#''Pyrocystis fusiformis,'' cells are available from Sunnyside Sea Farms at http://seafarms.com/html/products.html. Order the cultures in 10-mL tubes. The cultures they offer in plastic bags are also suitable. *Boxes with lids, opaque (3); each should be tall enough to one cup (with two test tubes inside) upright, with the lid tightly shut. If they are not, use bigger boxes. *Glue or tape *Plastic cups (3) *Masking tape *Permanent marker *Aluminum foil *Lamp with a fluorescent bulb *Nightlight or a flashlight *Lab notebook *Graph paper Experimental Procedure Note: You should begin this science fair project in the morning. Setting Up Your Cultures #When the cultures arrive, loosen the caps of the tubes. Follow any directions that are sent with the cultures to get them started. #Place a clean plastic cup in each box, near the middle. The cups will be used to keep the cultures upright. Glue or tape the cups in the boxes so that they won't fall over. Label the boxes as follows: ##''Dark'' ##''Light'' ##''Light/Dark'' #Label six of the tubes as follows: ##''A: Dark'' ##''B: Dark'' ##''C: Light'' ##''D: Light'' ##''E: Light/Dark'' ##''F: Light /Dark'' Note: If you've ordered the P. fusiformis cultures, you'll have more than six tubes and should label and add more replicates for each condition. #Wrap tubes A and B in aluminum foil. ##These will be kept in constant darkness. ##Place tubes A and B in the plastic cup in the box marked Dark. It's ok if the test tubes are not completely upright—leaning against the side of the cup is fine. ##Place the top on the Dark box and set it aside. #Place tubes C and D in the plastic cup in the box labeled Light. It's ok if the test tubes are not completely upright—leaning against the side of the cup is fine. #Put the lamp with the fluorescent bulb in an area where it can stay on 24 hours a day. #Place the Light box under the fluorescent lamp. #Turn on the lamp. ##Do not put the top on the Light box. ##These tubes will be kept in constant light. ##The cell cultures will be taken out of the light for brief periods of time, described later in the procedure, to determine if they will produce bioluminescence in response to mechanical stress. #The Dark box should now be placed next to the Light box, also under the lamp. ##Keep the boxes in the same area to minimize variations in temperature, etc. ##The boxes will both get equally heated since they are both under the lamp. ##Do not expose the cultures in the Dark box to any light. #Place tubes E and F in the cup in the box labeled Light/Dark, as follows: ##These cultures will be kept in darkness for part of the day, and in light for part of the day. ##Keep tubes E and F in the dark during the daylight hours. This will allow you to study their bioluminescence during your normal waking hours. ##Cover tubes E and F with aluminum foil in the morning. Choose a time that is convenient and wrap them at that time consistently. ##Place the wrapped tubes in the Light/Dark box and cover the box with the lid. ##Place the Light/Dark box near the other two boxes, also under the lamp. ##Unwrap tubes E and F in the evening. Unwrap them at the same times each day. Suggestion: Keep the cultures in the dark from 10:00 AM to 5:00 PM each day. ##Expose the Light/Dark tubes to the lamp light during the night. ##Re-wrap them in aluminum foil in the morning and place the lid back on the Light/Dark box. Making Observations Now that you have your Pyrocystis cultures set up, you can determine how the light and dark conditions affect their bioluminescence. The bioluminescence of the cultures can be viewed in a darkened room. #Create a scale for how bright the cultures glow. You will rank the brightness on a scale from 1 to 4, with 4 being the brightest. #Take the cultures into a dark room at the same times each day. You could have a dim light on, such as a nightlight or a flashlight, to help you work in the room. Keep the light several feet away from the cultures. Keep track of which tubes belong in which boxes. #To stimulate bioluminescence, tighten the cap on each tube, one at a time, and turn it upside down. You should see a clear blue light at the region where the bubble in the tube is moving through the culture. This region has the highest mechanical stress. Record your observations and brightness ranking in your lab notebook. #Test all of the cultures for bioluminescence. Record all observations and brightness rankings in your lab notebook. Try to treat each tube in the same way so that you can compare the relative amount of light produced. #Observe each of the cultures at least four times per day for five days. Keep track of dates, times, and all data in your lab notebook. #Make sure you observe the light/dark culture during both its light phase and its dark phase. #For example, if the light/dark cultures are in the dark from 10:00 AM to 5:00 PM, check the bioluminescence of the cultures (including the ones in constant darkness or light) at 8:00 AM, Noon, 4:00 PM, and 8:00 PM. Analyzing Your Data #Graph your data. ##Graph the time on the x-axis and the brightness score on the y-axis. ##Suggestion: Divide the x-axis into 24 hours, with noon in the middle. Mark the period of darkness for the light/dark cycle with a black rectangle under the x-axis. Add bars for the bioluminescent scores to the graph at the appropriate times. Use different colors for the all-light, all-dark, and light/dark cultures. #How does the varying light exposure affect the bioluminescence of the cells? #Repeat the procedure at least two more times so that you have three sets of data. Variations *Switch the cultures that have been in constant light (or constant darkness) to the light/dark cycle. How long does it take for their bioluminescent cycle to become fully adjusted to the new light/dark timing? For example, switch a culture that has been in constant darkness for 3 days to a dark (10:00 AM to 5:00 PM) /light (5:00 PM to 10:00 AM) cycle, and then record its bioluminescence (8:00 AM, Noon, 4:PM and 8:00 PM) for 7 days. Make a new graph for each day. *Look more carefully at when the cultures become bioluminescent in the light dark cycle. For example, if they are adjusted to a period of darkness from 10:00 AM to 5:00 PM, and light from 5:00 PM to 10:00 AM, check their bioluminescence every half hour between 8:00 AM and Noon, and every half hour between 3:00 PM and 7:PM. Graph your data. *Try other light/dark cycles and determine how the Pyrocystis cells respond. For example, 4 hours of darkness, followed by 20 hours of light, or 12 hours light/12 hours of darkness. How quickly do the cultures respond to a change in the light/dark cycle? *Borrow some beakers, a magnetic stir plate, and some stir bars from your school lab and investigate how the rate of rotation of the stir bar affects the amount of light produced. Set up an electronic measuring device to measure the light produced (see the Science Buddies project Measure Luminescence in Glow-in-the-Dark Objects for an example circuit). *Study the dinoflagellates under a microscope. Can you see any microscopic changes that follow a circadian rhythm? If so, what affect does altering the light/dark cycles have on these changes? Photos Category:Biotechnology Experiments